Induction of antibody- and cytotoxic T lymphocyte-mediated protective mucosal immunity, as well as systemic immunity, is important in HIV vaccine development. A key strategy will be to direct antigens to the potent specialized antigen presenting cells (APC), the dendritic cells (DC), in the common inductive sites of mucosal immunity. The major goal of this proposal is to target antigens to DC in mucosal tissues and skin. This will be done using sterically stabilized liposomes (SSL) targeted to DEC-205, a DC cell surface protein. Delivery of antigen to DC, using SSL conjugated with anti-DEC-205 monoclonal antibody NLDC- 145, will be quantified using in vitro assays: the approach is to devise a liposome formulation that has minimal uptake by macrophages via steric stabilization, and that has the ability to specifically bind to DC via antibody conjugation. In addition to antibody-mediated targeting to DC, the hemolysin from Listeria monocvtogenes, listeriolysin O (LLO), will be co-encapsulated inside the DC-immunotargeted liposomes. LLO co-encapsulated with antigenic protein inside liposomes can mediate the delivery of the protein into the cytosolic pathway of antigen presentation in macrophages. Thus, the LLO-containing liposomes offer a potential way of targeting antigens for both MHC class I- and MHC class II-restricted antigen presentation. Using mouse bone marrow-derived DC and fluorescence techniques, the applicants plan to measure both the specific enhancement of liposome uptake by DC via NLDC-145 conjugation and the delivery of a portion of liposomal content into DC cytosol. Additionally, a model antigen, ovalbumin (OVA), will be encapsulated inside liposomes to monitor the delivery to DC by the NLDC-145-conjugated, LLO-containing liposomes, and MHC class I- and MHC class II-restricted antigen presentation of OVA will be assayed. The optimal formulation developed in the in vitro studies will be tested in mice, localizing liposomes and their contents in DC of intestine and skin. Mice immunized with the DC-targeted SSL formulations will be tested for induction of mucosal immunity, by measuring OVA-specific antibody secretion into vaginal fluid. The liposome formulations proposed here could lead to the development of a safe yet efficient HIV vaccine that can elicit both mucosal and systemic protective immunity mediated by antibody and cytotoxic T lymphocyte responses.